Daniela Cesana

Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration

Insertional mutagenesis represents a major hurdle to gene therapy and necessitates sensitive preclinical genotoxicity assays. Cdkn2a−/− mice are susceptible to a broad range of cancer-triggering genetic lesions. We exploited hematopoietic stem cells from these tumor-prone mice to assess the oncogenicity of prototypical retroviral and lentiviral vectors. We transduced hematopoietic stem cells in matched clinically relevant conditions, and compared integration site selection and tumor development in transplanted mice. Retroviral vectors triggered dose-dependent acceleration of tumor onset contingent on long terminal repeat activity. Insertions at oncogenes and cell-cycle genes were enriched in early-onset tumors, indicating cooperation in tumorigenesis. In contrast, tumorigenesis was unaffected by lentiviral vectors and did not enrich for specific integrants, despite the higher integration load and robust expression of lentiviral vectors in all hematopoietic lineages. Our results validate a much-needed platform to assess vector safety and provide direct evidence that prototypical lentiviral vectors have low oncogenic potential, highlighting a major rationale for application to gene therapy.

The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy

gamma-Retroviral vectors (gammaRVs), which are commonly used in gene therapy, can trigger oncogenesis by insertional mutagenesis. Here, we have dissected the contribution of vector design and viral integration site selection (ISS) to oncogenesis using an in vivo genotoxicity assay based on transplantation of vector-transduced tumor-prone mouse hematopoietic stem/progenitor cells. By swapping genetic elements between gammaRV and lentiviral vectors (LVs), we have demonstrated that transcriptionally active long terminal repeats (LTRs) are major determinants of genotoxicity even when reconstituted in LVs and that self-inactivating (SIN) LTRs enhance the safety of gammaRVs. By comparing the genotoxicity of vectors with matched active LTRs, we were able to determine that substantially greater LV integration loads are required to approach the same oncogenic risk as gammaRVs. This difference in facilitating oncogenesis is likely to be explained by the observed preferential targeting of cancer genes by gammaRVs. This integration-site bias was intrinsic to gammaRVs, as it was also observed for SIN gammaRVs that lacked genotoxicity in our model. Our findings strongly support the use of SIN viral vector platforms and show that ISS can substantially modulate genotoxicity.

Site-specific integration and tailoring of cassette design for sustainable gene transfer

Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.

Comprehensive genomic access to vector integration in clinical gene therapy.

Retroviral vectors have induced subtle clonal skewing in many gene therapy patients and severe clonal proliferation and leukemia in some of them, emphasizing the need for comprehensive integration site analyses to assess the biosafety and genomic pharmacokinetics of vectors and clonal fate of gene-modified cells in vivo. Integration site analyses such as linear amplification–mediated PCR (LAM-PCR) require a restriction digest generating unevenly small fragments of the genome. Here we show that each restriction motif allows for identification of only a fraction of all genomic integrants, hampering the understanding and prediction of biological consequences after vector insertion. We developed a model to define genomic access to the viral integration site that provides optimal restriction motif combinations and minimizes the percentage of nonaccessible insertion loci. We introduce a new nonrestrictive LAM-PCR approach that has superior capabilities for comprehensive unbiased integration site retrieval in preclinical and clinical samples independent of restriction motifs and amplification inefficiency.

Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations

Gamma-retroviral/lentiviral vectors (γRV/LV) with self-inactivating (SIN) long terminal repeats (LTRs) and internal moderate cellular promoters pose a reduced risk of insertional mutagenesis when compared with vectors with active LTRs. Yet, in a recent LV-based clinical trial for β-thalassemia, vector integration within the HMGA2 gene induced the formation of an aberrantly spliced mRNA form that appeared to cause clonal dominance. Using a method that we developed, cDNA linear amplification-mediated PCR, in combination with high-throughput sequencing, we conducted a whole transcriptome analysis of chimeric LV-cellular fusion transcripts in transduced human lymphoblastoid cells and primary hematopoietic stem/progenitor cells. We observed a surprising abundance of read-through transcription originating outside and inside the provirus and identified the vector sequences contributing to the aberrant splicing process. We found that SIN LV has a sharply reduced propensity to engage in aberrant splicing compared with that of vectors carrying active LTRs. Moreover, by recoding the identified vector splice sites, we reduced residual read-through transcription and demonstrated an effective strategy for improving vectors. Characterization of the mechanisms and genetic features underlying vector-induced aberrant splicing will enable the generation of safer vectors, with low impact on the cellular transcriptome.

Uncovering and Dissecting the Genotoxicity of Self-inactivating Lentiviral Vectors In Vivo

Self-inactivating (SIN) lentiviral vectors (LV) have an excellent therapeutic potential as demonstrated in preclinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could still pose a significant risk in clinical applications. Taking advantage of novel in vivo genotoxicity assays, we tested a battery of LV constructs, including some with clinically relevant designs, and found that oncogene activation by promoter insertion is the most powerful mechanism of early vector-induced oncogenesis. SIN LVs disabled in their capacity to activate oncogenes by promoter insertion were less genotoxic and induced tumors by enhancer-mediated activation of oncogenes with efficiency that was proportional to the strength of the promoter used. On the other hand, when enhancer activity was reduced by using moderate promoters, oncogenesis by inactivation of tumor suppressor gene was revealed. This mechanism becomes predominant when the enhancer activity of the internal promoter is shielded by the presence of a synthetic chromatin insulator cassette. Our data provide both mechanistic insights and quantitative readouts of vector-mediated genotoxicity, allowing a relative ranking of different vectors according to these features, and inform current and future choices of vector design with increasing biosafety.

Lentiviral Vector Integration Profiles Differ in Rodent Postmitotic Tissues

Lentiviral vectors with self-inactivating (SIN) long terminal repeats (LTRs) are promising for safe and sustained transgene expression in dividing as well as quiescent cells. As genome organization and transcription substantially differs between actively dividing and postmitotic cells in vivo, we hypothesized that genomic vector integration preferences might be distinct between these biological states. We performed integration site (IS) analyses on mouse dividing cells (fibroblasts and hematopoietic progenitor cells (HPCs)) transduced ex vivo and postmitotic cells (eye and brain) transduced in vivo. As expected, integration in dividing cells occurred preferably into gene coding regions. In contrast, postmitotic cells showed a close to random frequency of integration into genes and gene spare long interspersed nuclear elements (LINE). Our studies on the potential mechanisms responsible for the detected differences of lentiviral integration suggest that the lowered expression level of Psip1 reduce the integration frequency in vivo into gene coding regions in postmitotic cells. The motif TGGAA might represent one of the factors for preferred lentiviral integration into mouse and rat Satellite DNA. These observations are highly relevant for the correct assessment of preclinical biosafety studies, indicating that lentiviral vectors are well suited for safe and effective clinical gene transfer into postmitotic tissues.

Lentiviral vector–based insertional mutagenesis identifies genes associated with liver cancer

Transposons and γ-retroviruses have been efficiently used as insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells and that hamper the identification of early cancer-driving events among bystander and progression-related events. We developed an insertional mutagenesis platform based on lentiviral vectors (LVVs) by which we could efficiently induce hepatocellular carcinoma (HCC) in three different mouse models. By virtue of the LVVs replication-deficient nature and broad genome-wide integration pattern, LVV-based insertional mutagenesis allowed identification of four previously unknown liver cancer–associated genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. The newly identified genes are likely to play a role in human cancer because they are upregulated, amplified and/or deleted in human HCCs and can predict clinical outcomes of patients.

Cyclosporin A and Rapamycin Relieve Distinct Lentiviral Restriction Blocks in Hematopoietic Stem and Progenitor Cells

Improving hematopoietic stem and progenitor cell (HSPC) permissiveness to HIV-derived lentiviral vectors (LVs) remains a challenge for the field of gene therapy as high vector doses and prolonged ex vivo culture are still required to achieve clinically relevant transduction levels. We report here that Cyclosporin A (CsA) and Rapamycin (Rapa) significantly improve LV gene transfer in human and murine HSPC. Both compounds increased LV but not gammaretroviral transduction and acted independently of calcineurin and autophagy. Improved gene transfer was achieved across all CD34(+) subpopulations, including in long-term SCID repopulating cells. Effects of CsA were specific of HSPC and opposite to its known impact on HIV replication. Mutating the Cyclophilin A binding pocket of the viral capsid (CA) further improved transduction in combination with CsA. Tracking of the LV genome fate revealed that CsA relieves a CA-dependent early block and increases integration, while Rapa acts early in LV infection independently of the viral CA. In agreement, only Rapa was able to improve transduction by an integrase-defective LV harboring wild-type CA. Overall, our findings pave the way for more efficient and sustainable LV gene therapy in human HSPCs and shed light on the multiple innate barriers specifically hampering LV transduction in these cells.

VISPA: a computational pipeline for the identification and analysis of genomic vector integration sites

The analysis of the genomic distribution of viral vector genomic integration sites is a key step in hematopoietic stem cell-based gene therapy applications, allowing to assess both the safety and the efficacy of the treatment and to study the basic aspects of hematopoiesis and stem cell biology. Identifying vector integration sites requires ad-hoc bioinformatics tools with stringent requirements in terms of computational efficiency, flexibility, and usability. We developed VISPA (Vector Integration Site Parallel Analysis), a pipeline for automated integration site identification and annotation based on a distributed environment with a simple Galaxy web interface. VISPA was successfully used for the bioinformatics analysis of the follow-up of two lentiviral vector-based hematopoietic stem-cell gene therapy clinical trials. Our pipeline provides a reliable and efficient tool to assess the safety and efficacy of integrating vectors in clinical settings.